1. Field of the Invention
The present invention relates to a method for observing a biological specimen.
This application is based on Japanese Patent Application No. 2007-330423, the content of which is incorporated herein by reference.
2. Description of Related Art
Recently, in research fields such as cellular biology or molecular cell biology, it has become necessary to observe biological cells by using green fluorescent protein (GFP) or a gene encoding luciferase, which is a bioluminescent enzyme, as a reporter of expression by labeling a specific portion or a functional protein inside the cells with a fluorescent label or a chemiluminescent label. Furthermore, techniques that use light for observing the conditions inside a biological specimen such as a small animal while living (in vivo) from outside the body have become important for, for example, medical investigation.
In general, for example, in the case of fluorescence observation of living cells into which a fluorescent protein is introduced, the intensity of a fluorescent image varies with time according to the expression state of the fluorescent protein or the activity of the cells. There are a variety of patterns of variation depending on the subjects being observed. For example, there are cases where the low intensity in the early stage of cell culturing increases with the expression of the fluorescent protein, cases where the intensity decreases with a decrease in the level of expression of the fluorescent protein, or cases where increases and decreases of intensity are repeated. These variations occur during luminescence observation of biological activity using living cells labeled with a luminescent label or during analysis of the position or size of a tumor by fluorescence observation of a biological specimen, such as a small animal, using a fluorescent protein. In these observations, it is important to acquire stable images that can be used for quantitative evaluation, over a period of time.
In one technique for acquiring stable images over a period of time, when images of fluorescence emitted from cells irradiated with excitation light are acquired at predetermined time intervals with a time-lapse image-acquisition system, it is known that the quantity of exposure light for acquiring a subsequent fluorescent image is determined using the previously acquired fluorescent image in order to prevent saturation of the fluorescent intensity (for example, Japanese Unexamined Patent Application, Publication No. 2005-214924) and that saturation of intensity is prevented by determining exposure conditions for acquiring a subsequent image based on the intensity values of cell images already captured, in each visual field (for example, Japanese Unexamined Patent Application, Publication No. 2006-317406).
In conventional methods, the optimum intensity value is obtained by changing the observation conditions, such as the exposure time, during the observation. With this approach, however, there is a disadvantage in that the quantitative properties tend to be lost when the observation conditions are changed. In particular, in the case where the quantitative properties are required to be maintained in long-period observation, it is undesirable to change the observation conditions, such as the exposure time or illumination power, during the observation.